Method for the modulation of acid-sphingomyelinase-related apoptosis

ABSTRACT

The present invention relates, first, to methods and compositions for the modulation of acid sphingomyelinase (ASM)-related processes, including apoptosis. Such apoptosis can include, but is not limited to, environmental stress-induced apoptosis such as, for example, ionizing radiation and/or chemotherapeutic agent-induced apoptosis. Apoptosis can be characterized by a cellular morphology comprising cellular condensation, nuclear condensation or zeiosis. The present invention further relates to methods for the identification of compounds which modulate (i.e., either increase or decrease) sensitivity to ASM-related processes, including apoptosis.

1. INTRODUCTION

The present invention relates, first, to methods and compositions forthe modulation of acid sphingomyelinase (ASM)-related processes,including apoptosis. Such apoptosis can include, but is not limited to,environmental stress-induced apoptosis such as, for example, ionizingradiation and/or chemotherapeutic agent-induced apoptosis. Apoptosis canbe characterized by a cellular morphology comprising cellularcondensation, nuclear condensation or zeiosis. The present inventionfurther relates to methods for the identification of compounds whichmodulate (i.e., either increase or decrease) sensitivity to ASM-relatedprocesses, including apoptosis.

2. BACKGROUND

The sphingomyelin pathway is a ubiquitous, evolutionarily conservedsignaling system initiated by hydrolysis of sphingomyelin to generatethe second messenger ceramide. Two forms of sphingomyelinase,distinguishable by the pH optima, are capable of initiating signaling.Acid sphingomyelinase (pH optimum 4.5-5.0) was originally identified asa lysosomal hydrolase required for turnover of cellular membranes (forreview see Kolesnick, R. N., 1991, Prog. Lipid Res. 30 1-38). However,Kronke and co-workers proposed that this enzyme was also targeted to theplasma membrane and signaled in response to activation of the 55 kD TNFreceptor (Wiegmann, K. et al., 1994, Cell 78:1005-1015). Activation ofacid sphingomyelinase has also now been associated with signaling viaFas, CD28 and the interleukin (IL)-1 receptor (Cifone, et al., 1993., J.Exp. Med. 177:1547-1552; Boucher, L.-M. et al., 1995, J. Exp. Med.181:2059-2068.; Liu. P. & Anderson, R. G. W., et al., 1995, J. Biol.Chem. 270:27179-27185. Human acid sphingomyelinase is the product of asingle gene, although alternative processing of the primary transcriptallows for the generation of multiple forms (Schuchman, E. H., et al.,1991, J. Biol. Chem. 266:8531-8539; Schuchman, E. H., et al., 1992,Genomics 12:197-205. Inherited mutations of the human acidsphingomyelinase gene lead to enzyme deficiency and the genetic disorderknown as Niemann Pick disease (NPD; Brady et al., R. O., et al., 1966,Proc. Natl. Acad. Sci. USA 55, 366-369.; Schneider, P. B. & Kennedy, E.P., 1967, J. Lipid Res. 8:202-209.

Neutral sphingomyelinase (pH optimum 7.4) was originally defined as aMg2⁺-dependent enzyme localized to the outer leaflet of the plasmamembrane (Rao, B. G. & Spence, M. W., 1976, J. Lipid Res. 17:506-515.;Yedger, S. & Gatt, S., 1976. Biochemistry 15:2570-2573. However, aMg2⁺-independent isoform of neutral sphingomyelinase which localizes tothe cytoplasm has recently been identified (Okazaki, T. et al., 1989, J.Biol. Chem. 264:19076-19080.; Okazaki, T. et al., 1994, J. Biol. Chem.269:4070-4077.). The neutral sphingomyelinase has not yet beencharacterized at the molecular level.

Neutral sphingomyelinase activation has been demonstrated in response tocellular stimulation with TNFa (Wiegmann, K. et al., 1994, Cell78:1005-1015) anti-Fas antibody (Tepper, C. G., et al., 1995, Proc.Natl. Acad. Sci. USA 92:8443-8447.; Cifone, M. G. et al., 1995, EMBO J.14:5859-5868.), and vitamin D (Okazaki, T. et al., 1989, J. Biol. Chem.264:19076-19080; Okazaki, T. et al., 1994, J. Biol. Chem.269:4070-4077.). It has also been suggested that neutralsphingomyelinase signals in response to IL-1b (Mathias, S. et al., 1993,Science 259:519-522) and ionizing radiation (Halmovitz-Friedman, A. etal., 1994, J. Exp. Med. 180:525-535.).

Although ceramide has been implicated as the second messenger for avariety of stress stimuli including TNFa, Fas ligand, ionizingradiation, heat shock, ultraviolet light and oxidative stress (Obeid, L.M. et al., 1993, Science 259:1769-1771.; Cifone, M. G. et al., 1993, J.Exp. Med. 177:1547-1552.; Jarvis, W. D., et al., 1994, Proc. Natl. Acad.Sci. USA 91:73-77; Fuks, Z., et al., 1994, Cancer Res. 54:2582-2590;Halmovitz-Friedman, A., et al., 1994, J. Exp. Med. 180:525-535.;Gulbins, E. et al., 1995, Immunity 2:341-351.; Verheij, M. et al., 1996,Nature 380:75-79; Jarvis, W. D. et al., 1996, Clin. Cancer Res. 2:1-6).Evidence for such speculation has been circumstantial (Verheij, M. etal., 1996, Nature 380:75-79; Hannun, Y. A. & Obeid, L. M, 1995, TrendsBiochem. Sci. 20:73-77). Definitive proof, therefore, that ceramidegeneration is a primary mediator of the apoptotic response is lacking.

3. SUMMARY OF THE INVENTION

The present invention relates, first, to methods and compositions forthe modulation of acid sphingomyelinase (ASM)-related processes,including apoptosis. Such apoptosis can include, but is not limited to,environmental stress-induced apoptosis such as, for example, ionizingradiation and/or chemotherapeutic agent-induced apoptosis. Apoptosis canbe characterized by a cellular morphology comprising cellularcondensation, nuclear condensation or zeiosis.

The present invention is based, in part, on the surprising discovery,described in the Example presented in Section 6, below, that acidsphingomyelinase activity is required for activation of stress-inducedapoptotic cellular pathways. Specifically, the data presented in theseExamples shows that ASM-deficient cell lines and ASM-deficient animalsare resistant to radiation-induced apoptosis. Thus, the data describedherein define, for the first time, an obligatory role for ceramidegeneration in signalling of stress-induced apoptosis.

The present invention further relates to methods for the identificationof compounds which modulate ASM-related processes, including apoptosis.“Modulation” as used herein, can refer, first, to an increase in thesensitivity of cells, specially neoplastic cells, to ASM-relatedprocesses, including apoptosis. Alternatively, “modulation” can refer toa decrease in the sensitivity of cells to ASM-related processes such asapoptosis; e.g., can refer to an increase in the cells' resistance toapoptosis.

Methods for the identification of compounds which increase a cell'ssensitivity to ASM-related processes such as apoptosis can be performedto identify targets and compounds which mimic ASM or act downstream ofASM in apoptotic pathways. Among the compounds and targets identifiedvia such identification methods are agents which can be utilized toincrease a neoplastic cell's sensitivity to apoptosis, thereby improvingthe clinical effects of anti-cell proliferative therapy, e.g. radiationand/or chemotherapeutic therapies.

Such methods can include, for example, a method comprising, firstcontacting an acid sphingomyelinase-deficient cell with a test compound,exposing the cell to a stress stimulus for a time sufficient to induceapoptosis in a cell exhibiting normal acid sphingomyelinase activity.Second, an acid sphingomyelinase-deficient cell is exposed, in theabsence of the test compound, to the stress stimulus for a timesufficient to induce apoptosis in a cell exhibiting normal acidsphingomyelinase activity. The exposed cells are monitored for thepresence of an apoptotic morphology, such that if the cell exposed tothe test compound exhibits a more severe apoptotic morphology, the testcompound represents a compound which increases a cell's sensitivity toacid sphingomyelinase-related apoptosis.

Alternatively, such methods for identifying a compound which increases acell's sensitivity to acid sphingomyelinase-related apoptosis can alsocomprise, first, contacting an acid sphingomyelinase-deficient cell witha test compound, and exposing the cell to a stress stimulus. Next, anacid sphingomyelinase-deficient cell is exposed, in the absence of thetest compound, to the stress stimulus. The levels of sphingomyelin andceramide present in the exposed cells are compared, such that if thelevel of sphingomyelin in the cell exposed in presence of test compoundis less than that of the cell exposed in the absence of the testcompound, or the level of ceramide in the cell exposed in the presenceof test compound is greater than that of the cell exposed in the absenceof test compound, the test compound represents a compound whichincreases a cell's sensitivity to acid sphingomyelinase-relatedapoptosis.

The cells utilized in the above-described methods for identifyingcompounds which increase a cell's sensitivity to ASM-related apoptosiscan be part of a genetically engineered nonhuman animal deficient forthe acid sphingomyelinase gene, such that the animal is exposed to thestress stimulus, either in the presence or absence of test compound.

Additionally, methods for the identification of compounds which decreasea cell's sensitivity to ASM-related processes such as apoptosis can beperformed. Such screens can identify additional targets in the apoptoticpathway which, like ASM, are necessary for stress-induced apoptosis tooccur. Further, such screens can identify compounds useful forminimizing the effects of stress-induced apoptosis, for example,apoptosis induced by radiation.

Such methods for identifying a compound which decreases a cell'ssensitivity to acid sphingomyelinase-related apoptosis can include, forexample, a method comprising, first, contacting a cell exhibiting acidsphingomyelinase activity with a test compound and exposing the cell toan apoptosis-inducing stress stimulus. Next, a cell which exhibits acidsphingomyelinase activity is exposed, in the absence of test compound,to the stress stimulus. The exposed cells are monitored for the presenceof an apoptotic morphology, such that if the cell exposed in thepresence of the test compound exhibits a less severe apoptoticmorphology, than the cell exposed in the absence of the test compound,the test compound represents a compound which decreases a cell'ssensitivity to acid sphingomyelinase-related apoptosis.

Such methods for identifying a compound which decreases a cell'ssensitivity to acid sphingomyelinase-related apoptosis, can alsoinclude, for example, a method comprising, first, contacting a cellexhibiting acid sphingomyelinase activity with a test compound, andexposing the cell to a stress stimulus. Next, a cell exhibiting acidsphingomyelinase activity is exposed, in the absence of test compound,to the stress stimulus. The levels of sphingomyelin and ceramide presentin the exposed-cells are compared such that if the level ofsphingomyelin in the cell exposed in the presence of test compound isgreater than that of the cell exposed in the absence of test compound,or the level of ceramide in the cell exposed in the presence of testcompound, is less than that of the cell exposed in the absence of testcompound, the test-compound represents a compound which decreases acell's sensitivity to acid sphingomyelinase-related apoptosis.

In the above-described methods for identifying compounds which decreasea cell's sensitivity to ASM-related apoptosis, the cells utilized can betransgenic cells comprising cells deficient in endogenous acidsphingomyelinase gene activity and containing a functional human acidsphingomyelinase transgene capable of expressing functional human acidsphingomyelinase. Further, such cells can be part of geneticallyengineered nonhuman animal deficient in endogenous acid sphingomyelinasegene activity and containing integrated in its cells a functional humanacid sphingomyelinase transgene capable of expressing functional humanacid sphingomyelinase.

In the above-described methods for identifying compounds which decreasea cell's sensitivity to ASM-related apoptosis, the cells utilized canalso be genetically engineered cells which exhibit a greater level ofacid sphingomyelinase activity than non-genetically engineered cell ofthe same type.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Normal lymphoblasts or MS1418 NPD lymphoblasts were irradiatedwith 20 Gy and incubated at 37° C. for the indicated times. Morphologicchanges of nuclear apoptosis were quantified by staining with theDNA-specific fluorochrome bisbenzimide. Cells with condensation ofchromatin, its compaction along the periphery of the nucleus, orsegmentation of the nucleus into three or more chromatin fragments wereconsidered apoptotic. A minimum of 500 cells were scored for theincidence of apoptosis. The data (mean+S.E.M.) represent two independentdeterminations from three separate experiments.

FIG. 1B. The dose-dependence of radiation-induced apoptosis was assessedin both types of lymphoblasts at 24 hours. Apoptotic cells werequantified as in FIG. 1A. The data (mean+S.E.M.) represent duplicatedeterminations from four separate experiments.

FIG. 1C. Normal or MS1271 NPD lymphoblasts were labeled to isotopicequilibrium with medium containing [3H]choline (1 mCi/ml), irradiatedwith 20 Gy, and incubated at 37° C. for the times indicated. Lipids wereextracted with chloroform:methanol:1 NHCl (100:100:1, v/v/v), subjectedto mild alkaline hydrolysis to remove glycerolipids, and sphingomyelin(SM) was resolved by thin-layer chromatography. Sphingomyelin levelswere determined by lipid scintillation spectrometry. The valuesrepresent mean+S.E.M. of independent triplicate determinations from twoseparate studies.

FIG. 1D. Unlabeled lymphoblasts were handled as in C and ceramide (Cer)levels were quantified by the diacylglycerol kinase assay as describedin experimental procedures. The values represent mean+S.E.M. ofindependent triplicate determinations from three separate studies withcontrol lymphoblasts and five experiments with MS1271 NPD lymphoblasts.

FIG. 2A. Retroviral transduction was performed by co-incubation for 48hours with amphotropic packaging cell lines which secrete acidsphingomyelinase-containing retrovirus as described in the experimentalprocedures. MS1271 NPD lymphoblasts transduced with the acidsphingomyelinase CDNA were incubated for 24 hours in serum-free mediumand then irradiated at 20 Gy. Ceramide levels were determined as in FIG.1D at the times indicated. The data (mean+S.E.M.) represent 2independent determinations from2 separate experiments. *p<0.05;**p<0.005.

FIGS. 2B,C. NPD lymphoblasts were infected with acidsphingomyelinase-containing retrovirus as in A and irradiated with 20Gy. Apoptosis was quantified by staining with bisbenzamide as in FIG. 1.Values represent the mean+S.E.M. of independent determinations from 3(MS1418) or 4 (MS1271) separate experiments.

FIGS. 3A,B. Male 129/SV (Acid sphingomyelinase (ASMase) +/+) andknock-out (ASMase −/−) mice received whole body radiation at 10 Gy 3(A)or at varying doses for 30 min 3(B), and at the indicated times weresacrificed by cervical dislocation. The lungs were dissected,homogenized in 8 volumes (w/v) of ice-cold PBS, and lipids wereextracted with chloroform:methanol (2:1, v/v). Ceramide levels weremeasured by fluorescence spectrometry after derivitization witho-phthaldehyde as described in experimental procedures. The valuesrepresent mean+S.E.M. of duplicate determinations from 2 separateexperiments.

FIG. 4. Radiation induces apoptosis in lungs of 129/SV (acidsphingomyelinase +/+) but not in the knock-out (acid sphingomyelinase−/−) mice. Lung specimens from 129/SV (ASMase +/+) and knock-out (ASMase−/−) mice were obtained 10 hours after exposure to 20 Gy whole bodyirradiation Tissues were fixed in formalin, paraffin-embedded, and 5micron sections were used for TUNEL assays. Apoptotic nuclei areidentified by brown-yellow staining, a product of the diaminobenzidinechromogen used. In contrast, normal nuclei stain blue due tocounterstaining with hematoxylin. Note the intense TUNEL signal in thenuclei of endothelial cells of small blood vessels and capillaries, andoccasionally in alveolar pneumocytes in the lung of the 129/SV mouse(left upper and lower panels). The majority of capillaries and smallblood vessels and all pneumocytes of the acid sphingomyelinase knock-outmice display negative TUNEL signals (right upper and lower panels).Original magnification: upper panel, x400; lower panel, x1000. Thisexperiment represents one of four similar studies.

FIG. 5A. Radiation induces a time-dependent increase in apoptosis in thethymus of C3H/HeJ mice. C3H/HeJ mice received whole body radiation at7.5 Gy and at the times indicated were sacrificed. Apoptosis in thymictissue was measured by TUNEL assay as described in FIG. 4. A minimum of1000 cells was scored for the incidence of apoptosis. The data(mean+range) represent duplicate determinations from one representativeof three separate experiments.

FIG. 5B. Quantitation of radiation-induced apoptosis in thymic andsplenic tissue from 129/SV (ASMase +/+) and knockout (ASMase −/−) mice.Two thousand cells were counted in four high power fields (x400).Numbers indicate apoptotic cells, and the percentage of apoptotic cellsis shown in arentheses. Differences between 129/SV and ASMase −/− wereevaluated by c2 and at each dose tested, P<0.001.

FIG. 6. Thymic samples from 129/SV (ASMase +/+) and knockout (ASMase−/−) mice were obtained 2.5 hours after exposure to 5 Gy whole bodyirradiation, and handled as in FIG. 4. Five micron sections were usedfor TUNEL assays, and apoptotic nuclei were identified as specified inFIG. 4. An intense TUNEL signal was noted in the nuclei of a proportionof thymocytes in the cortical region of 129/SV treated mice,occasionally forming small clusters of apoptotic cells (upper panel). Incertain instances, TUNEL staining was identified as a dark brown-to-bluereaction due to nuclear condensation of cells undergoing apoptosis. Thepercentage of positive TUNEL thymocytes was reduced in the acidsphingomyelinase knock-out mice, in which cluster formation was rare andonly scattered apoptotic cells were observed (lower panel). Originalmagnification: x400. This experiment represents one of three similarstudies.

FIG. 7. Radiation-induced apoptosis in thymic (upper panel) and lungtissue (lower panel) of C57Bl/6 (p53+/+) and p53 knock-out (p53−/−)mice. Thymic specimens from C57Bl/6 (p53+/+) and p53 knock-out mice wereobtained 10 hours after exposure to 20 Gy whole body irradiation, andhandled as in FIG. 6 (upper panels; original magnification: x400). Thelower panels show TUNEL stains of lung specimens obtained from the samemice (original magnification: x1000).

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Screening Assays forCompounds that Increase Sensitivity to ASM-Related Processes

Described in this Section are screening methods for identifyingcompounds that are capable of increasing a cell's sensitivity toASM-related processes, including apoptosis. Compounds identified veryscreens such as those described herein can be utilized, for example, asparts of methods for improving the clinical effects of radiationtherapy, as discussed, below, in Section 5.4.

As demonstrated in the Examples presented in Section 6, below,stress-induced apoptosis is dependent upon the presence of ASM activity,identifying ASM as an upstream regulator of the apoptotic response. Inorder to identify compounds which act increase a cell's sensitivity toapoptosis, assays-can, for example, be conducted on ASM-deficient cells,cell lines and/or animals to identify targets and compounds which mimicASM or act downstream of ASM in the apoptotic pathway.

Such assays can comprise exposing an ASM-deficient cell, cell line oranimal to a stress stimulus such as radiation, for example, ionizingradiation, in either the presence or absence of a test compound. Ininstances wherein the presence of the test compound is accompanied bythe appearance of apoptosis the test compound is to be considered onewhich increases a cell's sensitivity to ASM-related processes. It ispreferable that the apoptosis observed in the presence of the testcompound is more severe or more pronounced than that observed in'stress-exposed ASM-deficient cells, cell lines or animals not exposedto the test compound.

Such methods can include, for example, a method comprising, firstcontacting an acid sphingomyelinase-deficient cell with a test compound,exposing the cell to a stress stimulus for a time sufficient to induceapoptosis in a cell exhibiting normal acid sphingomyelinase activity.Second, an acid sphingomyelinase-deficient cell is exposed, in theabsence of the test compound, to the stress stimulus for a timesufficient to induce apoptosis in a cell exhibiting normal acidsphingomyelinase activity. The exposed cells are monitored for thepresence of an apoptotic morphology, such that if the cell exposed tothe test compound exhibits a more severe apoptotic morphology, the testcompound represents a compound which increases a cell's sensitivity toacid sphingomyelinase-related apoptosis.

Alternatively, such methods for identifying a compound which increases acell's sensitivity to acid sphingomyelinase-related apoptosis' can alsocomprise, first, contacting an acid sphingomyelinase-deficient cell witha test compound, and exposing the cell to a stress stimulus. Next, anacid sphingomyelinase-deficient cell is exposed, in the absence of thetest compound, to the stress stimulus. The levels of sphingomyelin andceramide present in the exposed cells are compared, such that if thelevel of sphingomyelin in the cell exposed in presence of test compoundis less than that of the cell exposed in the absence of the testcompound, or the level of ceramide in the cell exposed in the presenceof test compound is greater than that of the cell exposed in the absenceof test compound, the test compound represents a compound whichincreases a cell's sensitivity to acid sphingomyelinase-relatedapoptosis.

The cells utilized in the above-described methods for identifyingcompounds which increase a cell's sensitivity to ASM-related apoptosiscan be part of a genetically engineered nonhuman animal deficient forthe acid sphingomyelinase gene, such that the animal is exposed to thestress stimulus, either in the presence or absence of test compound.

Sphingomyelin and ceramide assays, as well as methods for the productionof acid sphingomyelinase deficient cells, cell lines and animals aredescribed, below, in Section 5.3.

5.2. Screening Assays for Compounds that Decrease Sensitivity toASM-Related Processes

Methods for the identification of compounds which decrease a cell'ssensitivity to ASM-related processes such as apoptosis are-described inthis Section. Such screens can identify targets in the apoptotic pathwayin addition to ASM which, like ASM, are necessary for stress-inducedapoptosis to occur. Further, such screens can identify compounds usefulfor minimizing the effects of stress-induced apoptosis, for example,radiation-induced apoptosis.

Such methods for identifying a compound which decreases a cell'ssensitivity to acid sphingomyelinase-related apoptosis can include, forexample, a method comprising, first, contacting a cell exhibiting acidsphingomyelinase activity with a test compound and exposing the cell toan apoptosis-inducing stress stimulus. Next, a cell which exhibits acidsphingomyelinase activity is exposed, in the absence of test compound,to the stress stimulus. The exposed cells are monitored for the presenceof an apoptotic morphology, such that if the cell exposed in thepresence of the test compound exhibits a less severe apoptoticmorphology, than the cell exposed in the absence of the test compound,the test compound represents a compound which decreases a cell'ssensitivity to acid sphingomyelinase-related apoptosis.

Such methods for identifying a compound which decreases a cell'ssensitivity to acid sphingomyelinase-related apoptosis, can alsoinclude, for example, a method comprising, first, contacting a cellexhibiting acid sphingomyelinase activity with a test compound, andexposing the cell to stress stimulus. Next, a cell exhibiting acidsphingomyelinase activity is exposed; in the absence of test compound,to the stress stimulus. The levels of sphingomyelin and ceramide presentin the exposed cells are compared such that if the level ofsphingomyelin in the cell exposed in the presence of test compound isgreater than that of the cell exposed in the absence of test compound,or the level of ceramide in the cell exposed in the presence of testcompound, is less than that of the cell exposed in the absence of testcompound, the test compound represents a compound which decreases acell's sensitivity to acid sphingomyelinase-related apoptosis.

In the above-described methods for identifying compounds which decreasea cell's sensitivity to ASM-related apoptosis, the cells utilized can betransgenic cells comprising cells deficient in endogenous acidsphingomyelinase gene activity and containing a functional human acidsphingomyelinase transgene capable of expressing functional human acidsphingomyelinase. Further, such cells can be part of a geneticallyengineered nonhuman animal deficient in endogenous acid sphingomyelinasegene activity and containing integrated in its cells a functional humanacid sphingomyelinase transgene capable of expressing functional humanacid sphingomyelinase.

In the above-described methods for identifying compounds which decreasea cell's sensitivity to ASM-related apoptosis, the cells utilized canalso be genetically engineered cells which exhibit a greater level ofacid sphingomyelinase activity than non-genetically engineered cells ofthe same type.

Sphingomyelin and ceramide assays, and methods for the production oftransgenic cells and animals comprising cells deficient in endogenousASM gene activity and containing a functional human ASM transgene, andgenetically engineered cells and animals which exhibit a greater levelof acid sphingomyelinase activity than non-genetically engineered cellsof the same type, are described, below, in Section 5.3.

5.3. Screening Assay Techniques 5.3.1. Apoptosis-Inducing Stimuli

The methods described in the Sections above can be used in identifyingcompounds which modulate a cell's sensitivity to ASM-related apoptosis.Among the apoptosis-inducing stimuli which can be tested areenvironmental stress agents, including but not limited to radiation,including ionizing radiation and/or chemotherapeutic agents.

Taking ionizing radiation as an example, cells and/or animals can beexposed to a radiation dosage range which will, preferably be between 0and 30 Gy, administered according to standard techniques well known tothose of skill in the art.

5.3.2. Apoptosis Assays

Cells can, in conjunction with the screening techniques described above,be assayed for apoptotic morphology using standard techniques well knownto those of skill in the art. Among the characteristics of apoptoticmorphology are cellular condensation, nuclear condensation, includingchromatin condensation, and the apoptotic characteristic plasma membraneruffling and bebbing referred to as “zeiosis” (Sanderson, C. J., 1982,in Mechanisms of Cell-Mediated Cytotoxicity, Clark, W. R. & Golstein,R., eds., Plenum Press, pp. 3-21; Godman, G. C. et al., 1075, J. CellBiol. 64:644-667).

For example, morphologic changes characteristic of nuclear apoptosis canbe assayed and quantified by stain the a DNA-specific fluorochrome suchas bis-benzimide (Hoechst-33258; Sigma according to standard methods(Bose, et al., 1995, Cell 82:405-414).

In vivo, apoptosis can be assayed via, for example, DNA terminaltransferase nick-end translation, or TUNEL assay, according to standardtechniques (Fuks, Z. et al., 1995, Cancer J. 1:62-72).

5.3.3. ASM, Sphingomyelin and Cetamide Assays

Acid sphingomeylinase activity, sphingomyelin levels and ceramide levelscan be measured via standard techniques well known to those of skill inthe art. See, for example, Maruyama, E. N. & Arima, M., 1989, J.Neurochem. 52:611-618; Quintern, L. E. et al., 1991, Meth. Enzymol.197:536-540; Wiegmann, K. et al., 1994, Cell 78:1005-1015, which areincorporated herein by reference in their entirety.

5.3.4. ASM Genes

Described in this Section are ASM-gene sequences which can be utilizedin conjunction with the production of ASM-deficient and transgeniccells, cell lines and animals such as those which can be utilized withthe screening techniques discussed, above.

The nucleotide sequence of the human ASM gene which can, for example, beutilized as the transgene in the production of transgenic cells andanimals deficient in endogenous ASM activity, but capable of expressinga transgenic ASM gene, is well known. See, for example, pending U.S.patent application Ser. No. 07/695,472, now U.S. Pat. No. 5,773287,which is incorporated herein by reference in its entirety.

The nucleotide sequence of the mouse ASM gene is also known and can beutilized in the production of, for example, ASM-deficient cells, cellline and animal (“knock outs”), as well as in the production of ASMoverexpressing cells, cell lines and genetically engineered animals.See, for example, Newrzella, D. & Stoffel, W., 1992, Hoppe-Seyler's Z.Bio. Chem. 373:1233-1238, which is incorporated herein by reference inits entirety.

In addition to the human and mouse ASM nucleotide sequences describedabove, ASM CDNA or gene sequences present in the same species and/orhomologs of the ASM gene present in other species can be identified andreadily isolated, without undue experimentation, by molecular biologicaltechniques well known in the art. Such sequences can, for example, beutilized in the production of knock and/or transgenic cells, cell linesand/or animals of species other than human or mouse.

For example, cDNA libraries synthesized from mRNA, or genomic DNAlibraries synthesized from genomic DNA, derived from the organism ofinterest can be screened by hybridization using either human or mouseASM sequences, as described above, as hybridization or amplificationprobes.

Screening can be by filter hybridization, using duplicate filters. Thelabeled probe can contain at least 15-30 base pairs of an ASM nucleotidesequence. The hybridization washing conditions used should be of a lowerstringency when the cDNA library is derived from an organism differentfrom the type of organism from which the labeled sequence was derived.

Low stringency conditions are well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.;and Ausubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y.

Alternatively, a labeled ASM nucleotide probe may be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions.

Further, an ASM gene homolog may be isolated from nucleic acid of theorganism of interest by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of ASM amino acidsequences encoded by the ASM sequences described above. The template forthe reaction may be CDNA obtained by reverse transcription of mRNAprepared from, for example, human or non-human cell lines or tissue,known or suspected to express an ASM gene allele.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an ASM gene. The PCRfragment may then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment may be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to isolate genomicclones via the screening of a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express the ASM gene). A reverse transcriptionreaction may be performed on the RNA using an oligonucleotide primerspecific for the most 5′ end of the amplified fragment for the primingof first strand synthesis. The resulting RNA/DNA hybrid may then be“tailed” with guanines using a standard terminal transferase reaction,the hybrid may be digested with RNAase H, and second strand synthesismay then be primed with a poly-C primer. Thus, cDNA sequences upstreamof the amplified fragment may easily be isolated. For a review ofcloning strategies which may be used, see e.g., Sambrook et al., 1989,supra.

With respect to transgenes, human ASM transgenes are preferred. DNAcontaining the nucleotide coding sequence for an entire human ASM geneproduct, or any portion capable of encoding a functional ASM geneproduct may be used to produce ASM transgenic cells, cell lines andanimals, such as, for example, those to be utilized as part of thescreening methods described, above.

Due to the degeneracy of the genetic code, DNA sequences which encodethe same or substantially same ASM gene product as that encoded by thehuman ASM nucleotide sequence described above can be utilized. The ASMnucleotide coding sequence used to produce the transgenic animals of theinvention can be regulated by human ASM promoter regulatory nucleotidesequences. Alternatively, such sequences can be regulated by promotersequences endogenous to the transgenes' host cells. Still further, anypromoter which is capable of driving the expression of the ASMtransgenic sequences in the transgenes' host cells can be utilized. Suchregulatory sequences will be well known to those of skill in the art,and can include both constitutive and inducible regulatory sequences.

5.3.5. ASM Knockout and Transgenic Animals and Cells

Cells, cell lines and animals deficient for ASM activity can, asdescribed above, be utilized as part of assays and screening techniquesfor the identification of compounds which modulate a cell's sensitivityto ASM-related apoptosis. The term “ASM-deficient”, as used herein,refers to cells, cell lines and/or animals which exhibit a lower levelof functional ASM activity than corresponding cells, or cell lines oranimals whose cells, contain two normal, wild type copies of the ASMgene. Preferably, “ASM-deficient” refers to an absence of detectablefunctional ASM activity.

A representative ASM-deficient, or “knockout” animal is a mouseASM-deficient animal. Such animals are well known to those of skill inthe art. See, for example, Horinouchi, K. et al., 1995, Nature Genetics10:288-293; and Otterbach, B. & Stoffel, W., 1995, Cell 81:1053-1061,both of which are incorporated herein by reference in their entirety.Techniques for generating additional ASM knockout cells, cell lines andanimals are described below.

Cells and cell lines deficient in ASM activity can be derived from ASMknockout animals, utilizing standard techniques well known to those ofskill in the art. Such animals may be used to derive a cell line whichmay be used as an assay substrate in culture. While primary cultures maybe utilized, the generation of continuous cell lines is preferred. Forexamples of techniques which may be used to derive a continuous cellline from the transgenic animals, see Small et al., 1985, Mol. CellBiol. 5:642-648. Such techniques for generating cells and cell lines canalso be utilized in the context of the transgenic and geneticallyengineered animals described below.

Further, ASM deficient cells can include cells taken from and cell linesderived from patient exhibiting Niemann-Pick disease, a disorder causedby an ASM deficiency. Such cells can include, for example, cellscontaining ASM mutations such as those described in U.S. patentapplication Ser. No. 08/250,740, now U.S. Pat. No. 5,686,240, which isincorporated herein by reference in its entirety. RepresentativeASM-deficient cell include MS1271 cells. MS1271 cells contain one alleleof the ΔR608 mutation and one allele of the R496L mutation (Levran etal., 1991, Proc. Natl. Acad. Sci. USA 88:37848-3752). AdditionalASM-deficient cells and cell lines can be generated using well knownrecombinant DNA techniques such as, for example, site-directedmutagenesis, to introduce mutations into ASM gene sequences which willdisrupt ASM activity.

Cells, cell lines and animals deficient for endogenous ASM activity and,further, containing an ASM transgene, preferably a human ASM transgene,capable of being expressed in the transgene, can also, as describedabove, be utilized as part of assays and screening techniques for theidentification of compounds which modulate a cell's sensitivity toASM-related apoptosis. Techniques for generating such transgenic cells,cell lines and animals are described below.

Further, genetically engineered cells, cell lines and animals whichexhibit a greater level of ASM activity than non-genetically engineeredcells of the same type can also, as described above, be utilized as partof assays and screening techniques for the identification of compoundswhich modulate a cell's sensitivity to ASM-related apoptosis. Techniquesfor generating such genetically engineered cells, cell lines and animalsare described below. Utilizing techniques such as those described above,for example, multiple copies of ASM transgenic or endogenous constructsmay be arranged within a vector may be stably introduced into thetransgenic founder animals to yield animals exhibiting overexpression ofASM.

ASM transgenic, ASM-deficient and ASM-overexpressing animals can begenerated using the ASM nucleotide sequences discussed, above, inSection 5.3.4. Such animals can be any species, including but notlimited to mice, rats, rabbits, guinea pigs, pigs, micro-pigs, andnon-human primates, e.g., baboons, squirrel monkeys and chimpanzees.

Any technique known in the art may be used to introduce a transgene(either a functional endogenous or heterologous transgene or,alternatively, an inactivating gene sequence) into animals to producethe founder lines of transgenic animals. Such techniques include, butare not limited to pronuclear microinjection (Hoppe, P. C. and Wagner,T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transferinto germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci.,USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson etal., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, MolCell. Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano etal., 1989, Cell 57:717-723); etc. For a review of such techniques, seeGordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, whichis incorporated by reference herein in its entirety).

As listed above, standard embryonal stem cell (ES) techniques can, forexample, be utilized for generation of ASM knockout, ASM-deficient andASM-overexpressing animals. ES cells can be obtained frompreimplantation embryos cultured in vitro (See, e.g., Evans, M. J. etal., 1981, Nature 292:154-156; Bradley, .O. et al., 1984, Nature309:255-258; Gossler et al., 1986, Proc. Natl. Acad. Sci. USA83:9065-9069; Robertson et al., 1986, Nature 322:445-448; Wood, S. A. etal., 1993, Proc. Natl. Acad. Sci. USA 90:4582-4584.)

Transgenes, which can include, for example, additional copies ofendogenous or heterologous ASM gene sequences (for, e.g., the productionof overexpressing ASM animals) and can, additionally contain transgenicASM gene sequences to be utilized as part of the production, e.g., ofASM transgenic animals deficient for endogenous ASM activity but capableof expressing an ASM transgene can efficiently be introduced into EScells by standard techniques such as DNA transfection orretroviral-mediated transduction. The resultant transformed ES cells canthereafter be combined with blastocysts from a non-human animal. Theintroduced ES cells thereafter colonize the embryo and contribute to thegerm line of a resulting chimeric animal (Jaenisch, R., 1988, Science240:1468-1474).

To accomplish ASM gene disruptions, either in the production of ASMknockout animals or as part of the production of ASM transgenic animalsdeficient for endogenous ASM activity, but containing a functional ASMtransgene, the technique of site-directed inactivation via genetargeting (Thomas, K. R. and Capecchi, M. R., 1987, Cell 51:503-512) andreview in Frohman et al., 1989, Cell 56:145-147; Cappecchi, 1989, Trendsin Genet. 5:70-76; Barribault et al., 1989, Mol. Biol. Med. 6:481-492;Wagner, 1990; EMBO J. 9:3025-3032; and Bradley et al., 1992,Bio/Technology.

Further, standard techniques such as, for example, homologousrecombination, coupled with the ASM sequences described, above, inSection 5.3.4, can be utilized to inactivate or alter any ASM geneticregion desired. A number of strategies can be utilized to detect orselect rate homologous recombinants. For example, PCR can be used toscreen pools of transformant cells for homologous insertion, followed byscreening of individual clones (Kim et al., 1988, Nucl. Acids Res.16:8887-8903; Kim et al., 1991, Gene 103:227-233). Alternatively, apositive genetic selection approach can be taken in which a marker geneis constructed which will only be active if homologous insertion occurs,allowing these recombinants to be selected directly (Sedivy et al.,1989, Proc. Natl. Acad. Sci. USA 86:227-231). Additionally, thepositive-negative approach (PNS) method can be utilized (Mansour et al.,1988, Nature 336:348-352; Capecchi, 1989, Science 244:1288-1292;Capecchi, 1989, Trends in Genet. 5:70-76). Utilizing the PNS method,nonhomologous recombinants are selected against by using the HerpesSimplex virus thymidine kinase (HSV-TK) gene and selecting against itsnonhomologous insertion with herpes drugs such as gancyclovir or FIAU.By such counter-selection, the number of homologous recombinants in thesurviving transformants is increased.

ES cells generated via techniques such as these, when introduced intothe germline of a nonhuman animal make possible the generation ofnon-mosaic, i.e., non-chimeric progeny. Such progeny will be referred toherein as founder animals. Once the founder animals are produced, theymay be bred, inbred, outbred, or crossbred to produce colonies of theparticular animal. Examples of such breeding strategies include but arenot limited to: outbreeding of founder animals with more than oneintegration site in order to establish separate lines; inbreeding ofseparate lines in order to produce compound transgenics that express thetransgene at higher levels because of the effects of additive expressionof each transgene; crossing of heterozygous transgenic mice to producemice homozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; breeding animals to different inbredgenetic backgrounds so as to examine effects of modifying alleles onexpression of the transgene and the neuropathological effects ofexpression.

6. EXAMPLE: ASM IS REQUIRED FOR RADIATION-INDUCED APOPTOSIS

The present studies address the role of ceramide generation via acidsphingomyelinase in induction of apoptosis using two separate geneticmodels. Lymphoblast cell lines from Niemann-Pick disease (NPD) patientsdemonstrated a defect in radiation-induced apoptosis which wasreversible upon restoration of acid sphingomyelinase activity. Defectsin radiation-induced apoptosis were also observed in tissues of acidsphingomyelinase knock-out mice, which contain physiologic levels ofneutral sphingomyelinase activity

6.6 MATERIALS AND METHODS Cell Culture

EBV-transformed lymphoblasts were established by standard techniques(Anderson, M. A. & Gusella, J. F., 1984, In Vitro 20:856-858.) from twoNPD patients, designated MS1271 and MS1418. MS1271 is an AshkenaziJewish Type B patient who is currently 11 years of age andneurologically intact. He carries one allele of the Type B mutation,AR608 (Levran, O., et al., 1991, J. Clin. Invest. 88:806-810), andanother of the mutation R496L (Levran, O. et al., 1991, Proc. Natl.Acad. Sci. USA 88:3748-3752). Cultured skin fibroblasts from thispatient have less than 3% of normal acid sphingomyelinase activity.MS1418 was derived from a 3 year old American boy of German ancestry whopresented with massive hepatosplenomegaly~and severe retardation. He wastreated for recurrent pneumonia and subsequently died when he was fiveyears of age. Cultured cells from patient MS1418 have <1% of normal acidsphingomyelinase activity. EBV-transformed lymphoblasts derived from NPDpatients or unaffected controls were maintained in a mixture of RPMI andDMEM media (4:1; v/v) containing 18% fetal calf serum (FBS) (Gibco BRL).Cells were grown at 37° C. in a 5% CO2 atmosphere. Cell number andviability were assessed by Trypan Blue exclusion analysis. Amphotropicpackaging cell lines which secrete acid sphingomyelinase-containingretrovirus (Yeyati, P. L. et al., 1995, Human Gene Therapy 6:975-983),were maintained in DMEM media with 10% FBS.

For gene transfer, retrovirus packaging cells were plated in 100 mmTranswell dishes (Costar), above the 0.45 mm membrane insert, at10-40×10⁶ cells per dish, and grown overnight. An equal number oflymphoblasts were layered beneath the Transwell insert on the followingday. After 48 hours of co-culture, infected lymphoblasts were removedfrom the dish and grown in fresh media. Expression of acidsphingomyelinase activity was maximal at 24-48 hours postinfection.Experiments were routinely performed at 24 hours post-infection.

Mice and Irradiation

Four to six week old male C3H/HeJ and C57BL/6 (p53+/+) mice werepurchased from the Jackson Laboratories (Bar Harbor, Maine), and 129/SVand p53 knock-out mice were purchased from Taconic Labs (Germantown,N.Y.). Acid sphingomyelinase knock-out mice were constructed asdescribed (Horinouchi, K. et al, 1995, Nature Genet. 10:288-293).Briefly, embryonic stem (ES) cells derived from 129/SV mice weretransfected with an acid sphingomyelinase replacement vector containinga neomycin expression cassette inserted into exon 2 of the acidsphingomyelinase gene. ES cell colonies containing the properly targetedacid sphingomyelinase sequences were obtained and then microinjectedinto blastocysts of C57BL/6 mice. The resulting chimeric mice were usedto generate the acid sphingomyelinase knock-out mouse colony. Thehomozygous acid sphingomyelinase knock-out phenotype is inherited as anautosomal recessive trait. For experiments, animals received whole bodyirradiation, delivered using a Cs-137 Irradiator (Shepherd Mark-I, Model68, SN643), at a dose rate of 270 cGy/min.

Lipid Studies

On the day of an experiment, cells were resuspended into media withoutserum (2×10⁶ cells/0.3 ml) and irradiated. Doses are indicated in eachfigure. Ceramide was quantified by the diacylglycerol kinase assay asdescribed (Dressler, & Kolesnick, R. N., 1990, J. Biol. Chem.256:14917-14921). Briefly, after irradiation, cells were incubated at37° C. for various lengths of time and extracted with 1 mlchloroform:methanol:1 N HCl (100:100:1, v/v/v). Lipids in the organicphase were dried under N2 and subjected to mild alkaline hydrolysis (0.1N methanolic KOH for 1 h at 37° C.) to remove glycerophospholipids.Samples were reextracted and lipids in the organic phase extract werequantified via the diacylglycerol kinase reaction.

For measurement of sphingomyelin levels, cells were labeled to isotopicequilibrium with [3H]choline (1 mCi/ml; Dupont NEN, specific activity79.2 Ci/mmol) for at least 3 cell doublings (Dressler, K. A. et al.,1992, Science 255:1715-1718.). Lipids were extracted as above andsphingomyelin was resolved by thin layer chromatography, usingchloroform:methanol:acetic acid:water (50:30:8:4) as solvent, identifiedby iodine vapor staining, and quantified by liquid scintillationcounting. Baseline sphingomyelin mass was verified by lipid phosphorousassay (Chen, J. P. S. et al., 1956, Anal. Chem. 28:1756-1758.).

Tissue ceramide content was determined by a modification of the methodused for amino acid analyses (Merrill, J.-A. H. et al., 1988, Anal.Biochem. 171:373-381.). After irradiation, animals were sacrificed bycervical dislocation and tissues were weighed and homogenized in 8 vol(w/v) of ice-cold PBS. Homogenate (0.4 ml) was transferred to 16×100 mmglass tubes and lipids were extracted with 2 ml of chloroform:methanol(2:1, v/v). Ceramide in the organic phase was measured after deacylationto sphingoid base and derivitization with o-phthaldehyde (OPA) asdescribed by Merrill et al., 1988. Briefly, aliquots of the organicphase (250 ml) were dried under N2, resuspended in 0.5 ml of 1 N KOH inmethanol and incubated for 1 hour at 100° C. to deacylate ceramide tofree sphingoid bases (Van Veldhoven, P. et al., 1989, Anal. Biochem183:177-189). Lipids were then dissolved in 50 ml methanol and mixedwith 50 ml of o-phthaldehyde reagent, which was prepared fresh daily bymixing 99 ml of 3% (w/v) boric acid in water (pH adjusted to 10.5 withKOH) and 1 ml of ethanol containing 50 mg of OPA (Sigma) and 50 ml of2-mercaptoethanol. After incubation for 5 min at room temperature, 500ml of methanol:5 mM potassium phosphate (pH 7.0) (90:10; v/v) was added,and the samples were clarified by brief centrifugation. Aliquots (20 ml)were quantified by reverse phase high performance liquid chromatography(HPLC) using a Nova Pak C18 column (60 Å, 4 μm, 3.9 mm×150 mm; Waters).Fluorescent lipids were eluted isocratically with methanol:5 mMpotassium phosphate, pH 7.0 (90:10; v/v) at a flow rate of 0.6 ml/minand detected by spectrofluorometer (excitation wavelength 340 nm,emission wavelength 455 nm). Ceramide levels were determined bycomparison to a concomitantly run standard curve of known amounts ofceramide (Type III: from bovine brain sphingomyelin; Sigma). The levelsof ceramide obtained by this procedure were similar to those obtained bythe diacylglycerol kinase assay.

Acid Sphingomyelinase Assay

Cells (1-2×10⁷) were pelleted (500 x g, 5 min, 4° C.), washed twice withice-cold PBS and resuspended (1×10⁶ cells/0.3 ml) into homogenizationbuffer (0.2% Triton X-100). Cells were disrupted with a Tenbroeck tissuehomogenizer (Bellco glass) and nuclei and debris were pelleted bycentrifugation at 800 x g for 5 min.

Acid sphingomyelinase activity was measured as described (Maruyama, E.N., & Arima, M., 1989, J. Neurochem. 52:611-618). Incubations contained30 mg of post-nuclear supernatant and 15 ml of sphingomyelin substrate(9 nmol sphingomyelin mixed with 0.9 ml of [¹⁴C]sphingomyelin, specificactivity 56 mCi/mmol; Amersham) in acid sphingomyelinase assay buffer(250 mM Na acetate pH 5.2, 1 mM EDTA, 0.1% Triton X-100). After 2 hoursat 37° C., 14C-phosphocholine was extracted with 200 ml ofchloroform:methanol (1:1 v/v) and 90 ml of H2O. Aliquots of the aqueousphase extract were quantified by liquid scintillation counting. Acidsphingomyelinase activity is expressed as nmols sphingomyelinhydrolyzed/mg protein/hr.

Apoptosis

Morphological changes of nuclear apoptosis were visualized by stainingwith the DNA-binding fluorochrome bisbenzimide (Hoechst-33258, Sigma) asdescribed (Bose, R., et al., 1995, Cell 82:405-414). Briefly,0.5-2.0×10⁶ cells were pelleted, washed once with PBS, and fixed in 500ml of 3% paraformaldehyde in PBS. Thereafter, cells were resuspendedinto 30 ml paraformaldehyde/PBS containing 16 mg/ml bisbenzimide.Aliquots were placed on glass slides, and evaluated by fluorescencemicroscopy (Olympus BH-2 fluorescence microscope with a BH2-DMU2UV DichMirror Cube filter). A minimum of 500 cells were scored for theincidence of apoptotic chromatin changes (condensation of chromatin, itscompaction along the periphery of the nucleus, and segmentation of thenucleus into greater than 3 fragments).

Apoptosis in vivo was assessed by the DNA terminal transferase nick-endtranslation method or TUNEL assay, as described (Fuks, Z., et al., 1994,Cancer Res. 54:2582-2590). Briefly, tissue specimens were fixedovernight in 4% buffered formaldehyde and embedded in paraffin blocks.Tissue sections (5 mm thick), adherent to polylysine-treated slides,were deparaffinized by heating at 90° C. for 10 minutes and then at 60°C. for 5 minutes. Tissue-mounted slides were first washed with 90% andthen 80% ethanol (3 minutes each) and rehydrated. The slides wereincubated in 10 mM Tris-HCl, pH 8 for 5 min, digested with 0.1% pepsin,rinsed in distilled water and treated with 3% H2O2 in PBS for 5 min at22° C. to inactivate endogenous peroxidase. After 3 washes in PBS, theslides were incubated for 15 min at 22° C. in buffer (140 mMNa-cacodylate, pH 7.2,30 mM Trizma base, 1 mM CoCl2) and then for 30minutes at 37° C. in reaction mixture (0.2 U/ml terminaldeoxynucleotidyl transferase, 2 nM biotin-11-dUTP, 100 mM Na-cacodylate,pH 7.0, 0.1 mM DTT, 0.05 mg/ml BSA and 2.5 mM CoCl2). The reaction wasstopped by transferring the slices to a bath of 300 mM NaCl, 30 mM Nacitrate for 15 min at 22° C. The slides were washed in PBS, blocked with2% human serum albumin in PBS for 10 min, re-washed and incubated withavidin-biotin peroxidase. After 30 minutes at 22° C., cells were stainedwith the chromogen 3,′diamonobenzidine tetrachloride and counterstainedwith hematoxylin. Nuclei of apoptotic cells appear brown and granular,while normal nuclei stain blue.

Statistical Analysis

Statistical analyses were performed by Student's test and Chi Squaretest.

6.2 RESULTS 6.2.1. Radiation Induces Apoptosis in Normal Lymphoblast butnot in Lymphoblasts from NPD Patients

FIG. 1A shows that exposure of EBV-transformed normal human lymphoblaststo a radiation dose of 20 Gy resulted in time-dependent apoptosis asdefined by morphologic changes of chromatin condensation and compaction,and nuclear segmentation. Apoptosis was detected by 8 hours and wasmaximal by 24 hours. As little as 1 Gy was effective and a peak effectwas achieved with 20 Gy (FIG. 1B). In contrast, the EBV-transformed NPDlymphoblast lines MS1418 (FIGS. 1A,B) and MS1271 did not demonstrate asignificant apoptotic response. The differences in the apoptoticresponse could not be attributed to altered cell proliferation, becausethe growth rates of NPD and normal lymphoblasts did not differappreciably.

6.2.2. Radiation Induces Ceramide Generation in Normal Lymphoblasts butNot in Lymphoblasts From NPD Patients

To determine whether these differences correlated with differences inceramide generation, normal and NPD lymphoblasts were exposed toionizing radiation and sphingomyelin hydrolysis to ceramide wasmeasured. Lymphoblasts from two normal individuals displayed acidsphingomyelinase activity of 6.0+0.2 nmol sphingomyelin hydrolyzed/mgprotein/hr (mean+range). The NPD lines, MS1418 and MS1271, expressedonly 2-3% residual acid sphingomyelinase activity of 0.19 and 0.13 nmolsphingomyelin hydrolyzed/mg protein/hr, respectively, consistent withpreviously published data (Suchi, M. et al., 1992, Proc. Natl. Acad.Sci. 89:3227-3231). FIG. 1C shows that a dose of 20 Gy induced a rapidreduction in sphingomyelin content in normal lymphoblasts from abaseline level of 440 pmol/106 cells. This effect was detected by 2 minand persisted for 30 minutes (p<0.001 vs. basal level). The reduction insphingomyelin content was accompanied by a near quantitative increase inceramide above a basal level of 180 pmol/106 vells (FIG. 1D). Ceramideelevation was detected by 1-2 min and was maximal at 15 min (p<0.001 vs.basal level). Consistent with the deficiency in acid sphingomyelinaseactivity, MS1271 lymphoblasts had elevated basal levels of sphingomyelinof 565 pmol/106 cells. These cells did not respond to 20 Gy radiation atany time between 0-30 min with a decrease in sphingomyelin (FIG. 1C) oran increase in ceramide content (FIG. 1D). Similar results were obtainedwith the NPD line MS1418, which exhibits a higher baseline sphingomyelincontent of 1,110 pmol/106 cells. It should be noted that the lack ofresponse was specific for deficiency of acid sphingomyelinase sincethese NPD lymphoblasts contain a normal level of neutralsphingomyelinase activity of 2.2 nmol sphingomyelin hydrolyzed/mgprotein/hr.

6.2.3. Retroviral Transfer of Acid Sphingomyelinase CDNA RestoresRadiation-Induced Ceraminde Generation and Apoptosis to NPD Lymphoblasts

To demonstrate a role for acid sphingomyelinase in induction ofapoptosis by ionizing radiation, acid sphingomyelinase activity wasrestored to the NPD lymphoblasts by retroviral transfer. Retroviraltransduction of the human acid sphingomyelinase cDNA into the NPDlymphoblast lines increased acid sphingomyelinase activity 17-fold inMS1418 cells to 3.24 nmol sphingomyelin hydrolyzed/mg protein/hr and8-fold in MS1271 cells to 1.03 nmol sphingomyelin hydrolyzed/mgprotein/hr. Routinely, expression was greater in line MS1418 than inMS1271. Introduction of the acid sphingomyelinase cDNA restoredradiation-induced ceramide generation to MS1271 (FIG. 2A) and MS1418cells, and apoptosis (FIG. 2B,C). In contrast, retroviral transductionof an irrelevant cDNA for the enzyme arylsulfatase did not restoreradiation-induced ceramide generation or apoptosis to MS1271 or MS1418cells.

6.2.4. Acid Sphingomyelinase Knock-Out Mice have a Defect inRadiation-Induced Apoptosis

In previous studies, it was reported that ionizing radiation inducedmarked apoptotic changes in the lungs of C3H/HeJ mice in vivo, asmeasured by the DNA-terminal transferase nick-end translation method(TUNEL) assay (Fuks, z. et al., 1994, Cancer Res. 54:2582-2590). Thewalls of pulmonary alveoli consist of an extensive network ofcapillaries, which allow for efficient gas exchange, and type I and IIpneumocytes for support. The prior investigations showed that theapoptotic response in the irradiated lung was largely confined tomicrovascular endothelial cells. This effect was detected by 6 hoursafter irradiation and was maximal by 10 hours. Thereafter, apoptoticcells were phagocytized by alveolar macrophages and could no longer bedetected by 24 hours.

To determine whether ceramide might play a role in radiation-inducedapoptosis in this mouse lung model, wild type C3H/HeJ mice were exposedto 10 Gy whole body radiation, and ceramide content was measured inextracts of lung tissue. Ceramide generation above a basal level of 280nmol/g lung tissue was detected as early as 5 minutes after irradiationand was maximal by 30 minutes. Thus ceramide generation preceded theonset of apoptosis. The effect of radiation on ceramide generation wasdose-dependent. As little as 7.5 Gy was effective and a maximal effectto 232% of control was demonstrated after 20 Gy at 30 min. This doserange is consistent with that previously shown to induce apoptosis inlungs of C3H/HeJ mice (Fuks, Z., et al., 1994, Cancer Res.54:2582-2590). It is believed that this is the first demonstration ofstress-induced ceramide generation in vivo.

To analyze the role of acid sphingomyelinase in radiation-inducedceramide generation and apoptosis in vivo, an acidsphingomyelinase-deficient mouse model was employed. Acidsphingomyelinase knock-out mice were generated by targeted disruption ofthe acid sphingomyelinase gene of 129/SV mice within exon 2 (Horinouchi,K. et al., 1995, Nature Genet. 10:288-293.). Homozygous acidsphingomyelinase knock-out mice appear normal at birth and developroutinely until about four months of age, when they begin to show signsof neurologic disease including ataxia, tremors and loss of appetite(Otterbach, B., & Stoffel, W., 1995, Cell 81:1053-61.; Horinouchi, K. etal., 1995, Nature Genet. 10:288-293.). Affected animals have nodetectable acid sphingomyelinase activity and accumulate sphingomyelinand cholesterol in various tissues as their disease progresses(Horinouchi, K. et al., 1995, Nature Genet. 10:288-293.). Acidsphingomyelinase knock-out mice were analyzed within 3 weeks of weaning,a time at which no disease manifestations are detected.

Similar to C3H/HeJ mice, radiation induced time- and dose-dependentceramide generation above a basal level of 225 nmol/g tissue in thelungs of the wild type 129/SV mice (FIG. 3). However, radiation did notincrease ceramide content in the lungs of the acid sphingomyelinaseknock-out mice. Exposure to 20 Gy induced extensive apoptosis in thelungs of wild type 129/SV mice after 10 hours (FIG. 4; left upperpanel). Higher magnification of the these tissue specimens demonstratedthat apoptosis occurred primarily in the endothelium (FIG. 4; left lowerpanel). In contrast, acid sphingomyelinase-deficient mice did notexhibit significant pulmonary apoptosis in response to 20 Gy (FIG. 4C;right panel). Increasing the radiation dose up to 30 Gy failed togenerate an apoptotic response in the lungs of the acidsphingomyelinase-deficient mice.

The effect of radiation to induce apoptosis was also evaluated in thymicand splenic tissue from 129/SV and C3H/HeJ mice. Both strains yieldedidentical results. Apoptosis occurred more rapidly in thymic than lungtissue, and the response was biphasic. A rapid phase, detected by 1 hourwith 7.5 Gy, peaked at 3 hours, and was followed by a slower response(FIG. 5A). As little as 2 Gy was effective and a maximal effect wasachieved with 10 Gy. As compared to the lung, the acid sphingomyelinaseknock-out mice demonstrated a less comprehensive defect inradiation-induced apoptosis in thymic tissue. Unirradiated thymic tissuefrom normal and knock-out animals manifested a baseline 4-5% incidenceof apoptosis. FIG. 6 shows that a dose of 5 Gy induced substantialapoptosis in the thymic cortex of 129/SV mice at 2.5 hours afterirradiation. Many of the apoptotic cells formed clusters of 5-8 cellssurrounded by normal appearing cells. In contrast, minimal apoptosis wasdetected in the thymic cortex of the acid sphingomyelinase knock-outmice. When 2000 129/SV thymic cells were counted in four high-powerfields (FIG. 5B), a total of 652 (33%) showed apoptotic changes. Incontrast, only 346 (17%) of 2000 cells in the thymic cortex of the acidsphingomyelinase knock-out mouse were apoptotic (p<0.0001).Statistically significant differences in thymic apoptosis were alsoobserved between 129/SV and knock-out mice at 4 Gy and 7.5 Gy (FIG. 6C).After 4 hours, there was a rapid increase in the incidence of apoptosisin both strains. A diffuse pattern gradually developed throughout thethymic cortex and detectable differences diminished, perhaps due toovercrowding by the late responding apoptotic cells. Differences were nolonger detected by 10 hours. Qualitatively similar results were observedin the spleen (FIG. 5B).

6.2.5. Comparison of p53 and Acid Syhingomyelinase Knock-Out Mice

Because of the variation in tissue responses of the acidsphingomyelinase knock-out mice, p53 knockout mice, also known to bedefective in radiation-induced apoptosis, were studied. FIG. 7 showsapoptotic responses to 20 Gy in wild type C57Bl/6 mice and the p53knock-out mice derived from this strain. Both the cortex (left upperpanel) and medulla of the wild type thymus demonstrated diffuseapoptosis at 10 hours. In contrast, only 13% of the thymic cells of thep53 knock-out mice expressed apoptotic changes (right upper panel),appearing mostly in clusters of 5-8 cells. Increasing the radiation doseto 30 Gy failed to increase the apoptotic response in the thymus of thep53-deficient mice. These in vivo studies agree with previous ex vivostudies, which reported a 10-20% incidence of radiation-inducedapoptosis in thymocytes derived from p53 knock-out mice (Strasser, A. etal., 1994, Cell 79:329-339; Lowe, S. W., et al., 1993. p53-Dependentapoptosis modulates the cytotoxicity of anticancer agents. Cell74:957-967.).

In contrast to the thymus, lungs of p53-deficient mice (lower rightpanel) exhibited a normal apoptotic response at 10 hours after exposureto 20 Gy. The effect appeared identical to that observed in wild-typeC57Bl/6 mice (lower left panel), and in 129/SV (FIG. 4) and C3H/HeJ mice(Fuks, Z., et al., 1994, Cancer Res. 54:2582-2590). It should again benoted that the lungs of acid sphingomyelinase-deficient mice did notundergo apoptosis in response to radiation (FIG. 4).

To explore the sensitivity of other p53-deficient tissues toradiation-induced apoptosis, knock-out mice were exposed to whole-bodyradiation (20 Gy). Apoptotic responses were observed at 10 hours in thepleura, endocardium, pericardium, and the germinal centers of thespleen. Apoptotic responses were not observed in liver, kidney, brain,skin, the myocardium and striated muscle. This pattern of tissueresponse was identical to that observed in C57Bl/6, 129/SV and C3H/HeJmice when exposed to a similar radiation dose. However, the radiationresponse in acid sphingomyelinase knock-out mice differed. These micedid not develop apoptosis in endocardium, pericardium or pleura.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

What is claimed is:
 1. A method for identifying a compound whichincreases a cell's sensitivity to acid sphingomyelinase-relatedapoptosis, comprising: (a) contacting an acid sphingomyelinase-deficientcell with a test compound; (b) exposing the cell to a radiation stressstimulus for a time sufficient to induce apoptosis in a cell exhibitingnormal acid sphingomyelinase activity; (c) exposing an acidsphingomyelinase-deficient cell, in the absence of the test compound, tothe radiation stress stimulus for a time sufficient to induce apoptosisin a cell exhibiting normal acid sphingomyelinase activity; and (d)monitoring the exposed cells of steps (b) and (c) for the presence of anapoptotic morphology, such that if the cell from step (b) exhibits amore severe apoptotic morphology, than that of the cell from step (c)the test compound represents a compound which increases a cell'ssensitivity to acid sphingomyelinase-related apoptosis.
 2. A method foridentifying a compound which increases a cell's sensitivity to acidsphingomyelinase-related apoptosis, comprising: (a) exposing acidsphingomyelinase-deficient cells, wherein the cells are part of celllines or a genetically engineered nonhuman animal deficient for the acidsphingomyelinase gene, in the presence or the absence of a testcompound, to a radiation stress stimulus for a time sufficient to induceapoptosis in a cell exhibiting normal acid sphingomyelinase activity;and (b) monitoring the exposed cells of step (a) for the presence of anapoptotic morphology, such that if the cells treated with the testcompound exhibit a more severe apoptotic morphology than that of thecells not treated with the test compound, the test compound represents acompound which increases a cell's sensitivity to acidsphingomyelinase-related apoptosis.
 3. The method of claims 1 or 2wherein the apoptotic morphology comprises cellular condensation,nuclear condensation or zeiosis.
 4. A method for identifying a compoundwhich increases a cell's sensitivity to acid sphingomyelinase-relatedapoptosis, comprising: (a) contacting an acid sphingomyelinase-deficientcell with a test compound; (b) exposing the cell to a radiation stressstimulus; (c) exposing an acid sphingomyelinase-deficient cell, in theabsence of the test compound, to the radiation stress stimulus; and (d)comparing the levels of sphingomyelin and ceramide present in theexposed cell of step (b) to the levels present in the exposed cell ofstep (c), such that if the level of sphingomyelin in the cell of step(b) is less than that of the cell of step (c)., or the level of ceramidein the cell of step (b) is greater than that of the cell in step (c),the test compound represents-a compound which increases a:cell-'s-sensitivity to acid sphingomyelinase-related apoptosis.
 5. Amethod for identifying a compound which decreases a cell's sensitivityto acid sphingomyelinase-related apoptosis, comprising: (a) contacting acell exhibiting acid sphingomyelinase activity with a test compound; (b)exposing the cell to a radiation stress stimulus; (c) exposing a cellexhibiting acid sphingomyelinase activity to the radiation stressstimulus, in the absence of the test compound; and (d) comparing thelevels of sphingomyelin and ceramide present in the exposed cell of step(b) to the levels present in the exposed cell of step (c), such that ifthe level of sphingomyelin in the cell of step (b) is greater than thatof the cell of step (c), or the level of ceramide in the cell of step(b) is less than that of the cell in step (c), the test compoundrepresents a compound which decreases a cell's sensitivity to acidsphingomyelinase-related apoptosis.
 6. A method for identifying acompound which increases a cell's sensitivity to acidsphingomyelinase-related apoptosis, comprising: (a) exposing acidsphingomyelinase-deficient cells, wherein the cells are part of celllines or a genetically engineered nonhuman animal deficient for the acidsphingomyelinase gene, in the presence or the absence of a testcompound, to a radiation stress stimulus for a time sufficient to induceapoptosis in a cell exhibiting normal acid sphingomyelinase activity;and (b) comparing the levels of sphingomyelin and ceramide present incells treated with test compound to cells untreated with the testcompound, such that if the level of sphingomyelin in the cells treatedwith the test compound is less than that of cells not treated with thetest compound, or the level of ceramide in cells treated with the testcompound is greater than in cells not treated with the test compound,the test compound represents a compound which increases a cell'ssensitivity to acid sphingomyelinase-related apoptosis.
 7. A method foridentifying a compound which decreases a cell's sensitivity to acidsphingomyelinase-related apoptosis, comprising, (a) exposing transgeniccells, comprised of cells deficient in endogenous acid sphingomyelinasegene activity that contain a functional human acid sphingomyelinase genecapable of expressing functional human acid sphingomyelinase, to aradiation stress stimulus in the presence or absence of a test compound;and (b) comparing the levels of sphingomyelin and ceramide present incells treated with test compound to cells not treated with the testcompound, such that if the level of sphingomyelin in cells treated withthe test compound is greater than in cells not treated with the testcompound, or the level of ceramide in cells treated with the testcompound is less than that of cells not treated with the test compound,the test compound represents a compound which decreases a cell'ssensitivity to acid sphingomyelinase-related apoptosis.
 8. The method ofclaim 7 wherein the cell is part of a genetically engineered nonhumananimal deficient in endogenous acid sphingomyelinase gene activity andcontaining integrated in its cells a functional human acidsphingomyelinase transgene capable of expressing functional human acidsphingomyelinase.
 9. A method for identifying a compound which decreasesa cell's sensitivity to acid sphingomyelinase-related apoptosis,comprising, (a) exposing cells, wherein the cells are geneticallyengineered cells that exhibit a greater level of acid sphingomyelinaseactivity than non-genetically engineered cells of the same type, to aradiation stress stimulus in the presence or absence of a test compound;and (b) comparing the levels of sphingomyelin and ceramide present incells treated with the test compound to cells not treated with the testcompound, such that if the level of sphingomyelin in cells treated withthe test compound is greater than in cells not treated with the testcompound, or the level of ceramide in cells treated with the testcompound is less than that of cells not treated with test compound, thetest compound represents a compound which decreases a cell's sensitivityto acid sphingomyelinase-related apoptosis.